In 2006, data from the U.S. Health Cost &Utilization Project revealed over one million orthopedic-related procedures, at a cost in excess of 7 billion dollars making skeletal repair using tissue engineering an attractive target. The ability of adipose-derived stromal cells (ASCs) to differentiate into bone suggests that they may be used to fulfill the mounting needs of patients with genetic disorders, degenerative diseases, and traumatic or post-surgical tissue deficits of the skeleton. Though similar techniques can be performed with bone marrow cells and embryonic stem cells, adipose cells are easier to obtain in large quantities and lack the ethical concerns. The ultimate translational goal would be the ability within one visit to the operating room to harvest adipose tissue, obtain sufficient numbers of human ASCs, isolate those cells with a greater osteogenic potential and implant these cells into the skeletal defect. The proposal below will facilitate the development of a novel means to promote rapid and robust bone formation in patients with skeletal defects. The central hypothesis is that human adipose derived stromal cells represent a readily available population of cells with which to design cell-based therapies for skeletal regeneration. In the first specific aim, a human ASC subpopulation with enhanced osteogenic capacity in vitro will be identified. Preliminary studies have shown the cell surface marker CD105 to have an increased osteogenic potential in mice and this same marker will be used to identify humans cells with increased osteogenic capabilities. Human adipose tissue will be harvested using standard liposuction techniques. Human ASCs with enhanced osteogenic potential will be isolated based on cell surface markers (CD105high and CD105low) using fluorescence activated cell sorting (FACS). The osteogenic potential of these two subpopulations will be analyzed using QT-PCR and Alizarin red staining. In the second specific aim, skeletal healing will be accelerated in vivo by implanting the subpopulation of human ASCs with enhanced osteogenic potential from Specific Aim #1 onto a skeletal defect using a PLGA scaffold. Osseous healing will be assessed and quantified using microcomputed tomography (microCT), detailed histology, histomorphometry and fluorescent in situ hybridization(FISH). Data obtained from these aims will demonstrate an innovative strategy to repair and regenerate bone that has been affected by trauma, disease, surgery or malformations which is consistent with the NIH mission to reduce the burdens of disability.